Engineered Anisotropic and Vascularized Human Cardiac Patch Project Summary Currently there are no therapies to effectively reverse cardiac injury following myocardial infarction. Tissue Engineering holds promise for the regeneration of heart tissue through an engineered cardiac patch. Unfortunately, pervious efforts have failed to achieve a cardiac patch with an effective engraftment rate and regenerative efficacy. Human pluripotent stem cell-derived cardiac fibroblasts (hPSCs-CFs) have the potential to provide an unlimited supply of cardiac tissue-specific extracellular matrix (ECM), which could be organized into nanofibers and serve as a universal scaffold to direct the anisotropic orientation of cells and engineered microvessels into a biomimetic hierarchical structure. Human mesenchymal stem cells (hMSCs) are immunomodulatory and effective in promoting myocardial regeneration, and can function as pericytes to mature and stabilize microvessels constructed by endothelial cells. Human pluripotent stem cell-derived cardiomyocytes (hPSCs-CMs) could orchestrate contractile synchrony between the transplanted cardiac tissue and the underlying myocardium. We hypothesize that the combination of hPSCs-CF derived cardiac tissue- specific ECM nanofibers with hMSCs and capillary-like microvessels will significantly enhance the anisotropic cardiac tissue engraftment and effectively promote myocardial regeneration. The objective of the proposed project is to biomimetically engineer an anisotropic cardiac patch containing aligned cardiac tissue-specific nanofibrous ECM, dense and oriented capillary-like microvessels, contractile CMs, and regeneration-promoting hMSCs within a short time. With a strong team comprising of a well-established tissue engineer, a world leading cardiovascular physician-scientist, a very experienced cardiovascular physiologist, an expert in electrophysiology and optical mapping, and an experienced biostatistician, we will pursue the specific aims: I. Derive an aligned and uniform cardiac-specific nanofibrous ECM scaffold from hPSC-CFs. II. Develop an anisotropic, vascularized and contractile cardiac patch representative of native myocardium. III. Evaluate the anastomosis, engraftment and regeneration efficacy of the prevascualrized cardiac patch in a rat MI model.